Simple and Fast Maximally Deuterated Control (maxD) Preparation for Hydrogen–Deuterium Exchange Mass Spectrometry Experiments

During the analysis steps of hydrogen–deuterium
exchange
(HDX) mass spectrometry (MS), there is an unavoidable loss of deuterons,
or back-exchange. Understanding back-exchange is necessary to correct
for loss during analysis, to calculate the absolute amount of exchange,
and to ensure that deuterium recovery is as high as possible during
liquid chromatography (LC)–MS. Back-exchange can be measured
and corrected for using a maximally deuterated species (here called
maxD), in which the protein is deuterated at positions and analyzed
with the same buffer components, %D2O, quenching conditions,
and LC–MS parameters used during the analysis of other labeled
samples. Here, we describe a robust and broadly applicable protocol,
using denaturation followed by deuteration, to prepare a maxD control
sample in ∼40 min for nonmembrane proteins. The protocol was
evaluated with a number of proteins that varied in both size and folded
structure. The relative fractional uptake and level of back-exchange
with this protocol were both equivalent to those obtained with earlier
protocols that either require much more time or require isolation
of peptic peptides prior to deuteration. Placing strong denaturation
first in the protocol allowed for maximum deuteration in a short time
(∼10 min) with equal or more deuteration found in other methods.
The absence of high temperatures and low pH during the deuteration
step limited protein aggregation. This high-performance, fast, and
easy-to-perform protocol should enhance routine preparation of maxD
controls.